Introduction

Acute myeloid leukemia (AML) is characterized by expansion of myeloid blasts with suppression of normal hematopoiesis. Cytogenetic and molecular studies have defined AML as a heterogeneous disease comprised of genetically distinct clonal disorders. Whereas recurring chromosomal translocations are a hallmark of AML, these genetic lesions are thought to cooperate with mutations in genes that encode growth factor receptors and downstream effectors.¹ FLT3 and CKIT encode class III receptor tyrosine kinases that activate Ras and other signaling cascades.² Dominant activating mutations in FLT3, CKIT, NRAS, and KRAS have been reported in up to 50% of patients with AML.^{2, 3, 4} Uncovering other mutations that perturb the RTK/Ras signaling pathways could further illuminate the biology of AML, provide new independent prognostic markers, and/or identify new therapeutic targets.

The PTPN11 gene encodes SHP-2, a nonreceptor protein tyrosine phosphatase that relays signals from activated growth factor receptors to Ras and other signaling molecules.⁵ Germline mutations in PTPN11 are a major cause of Noonan syndrome (NS).^{6, 7} Children with NS are at increased risk of developing juvenile myelomonocytic leukemia (JMML), a relentless myeloproliferative disorder characterized by overproduction of myelomonocytic cells that infiltrate spleen, liver, and skin, among other organs.^{8, 9, 10} Most children with JMML are boys.^{8, 9, 10} We and others have recently reported that up to 35% of patients with JMML harbor missense PTPN11 mutations, which are largely exclusive of RAS and NF1 alterations.^{11, 12} All of these mutations are predicted to activate SHP-2 phosphatase activity by disrupting the inhibitory interaction between the N-SH2 and PTP domains of the protein. Although the biochemical data are inconclusive, genetic studies suggest that the hyperactive PTPN11 mutations found in JMML contribute to leukemia growth by deregulating the Ras pathway.^{11, 12}

Based on the existence of NRAS and KRAS mutations in AML and JMML, we reasoned that mutations in PTPN11 might also occur in both diseases. To address this question, we screened a well-characterized cohort of AML specimens from children enrolled on Children's Cancer Group (CCG) clinical trials 2941 and 2961 for mutations in PTPN11 and correlated these data with clinical and molecular data.

Materials and methods

Patients and therapy

Bone marrow specimens from 298 patients with de novo AML registered on protocols CCG 2941 and 2961 were included in this study. A total of 278 specimens had adequate DNA for analysis. The diagnosis of AML was made according to FAB classification and confirmed by central review. The treatments delivered on CCG 2941 and 2961 have been previously detailed.^{13, 14} The study was approved by the UCSF Committee on Human Research and the CCG Myeloid Biology Committee.

Mutation detection

DNA was extracted from cryopreserved bone marrow cells using PureGene reagents (Gentra Systems Inc, Minneapolis, MN, USA). Methods for the mutational analysis for PTPN11, NRAS, and FLT3 internal tandem duplications (ITD) and FLT3 activating loop mutations (ALM) have been previously described.^{2, 7, 12, 15} Based on the locations of mutations in NS and JMML, exons 3, 4, 5, 6, 7, 8, 11, 12, and 13 of PTPN11 were screened.

Statistical methods

Data from CCG 2941 (through March 2002) and 2961 (through January 2004) were used to compare patients who had samples analyzed for PTPN11 mutations with those who did not, and to assess the characteristics of patients with and without PTPN11 mutations. The significance of observed differences was tested using the ² and Fisher exact tests. For continuous data, the Mann–Whitney test was used to compare the medians of distributions. Patients who were lost to follow-up were censored at their last known point of study, with a cutoff of September 2001 (CCG 2941) or July 2003 (CCG 2961). Actuarial estimates of overall survival (OS) and event-free survival (EFS) from study entry and disease-free survival (DFS), defined as the time from achieving remission to marrow relapse or death, were estimated using the Kaplan–Meier method.¹⁶ Corresponding standard errors were calculated using Greenwood's formula.¹⁷ Differences in OS, EFS, and DFS were tested for significance using the log rank statistic.¹⁸

Results and discussion

To determine whether our study population was representative of the entire CCG-2941/2961 population (n=988), the clinical characteristics and outcome of the 278 analyzed patients were compared to the 710 patients that were not studied. There were no significant differences with respect to age, sex, race, presenting white count, hemoglobin, platelet count, or FAB morphology. Cytogenetic information was available on 549 cases (56%), including 170 that we analyzed for PTPN11 mutations. There were no significant differences with respect to the distribution of previously defined good, poor, or standard risk cytogenetics,¹⁹ survival, or event-free survival between the study population and the rest of the patient cohort (data not shown).

We identified 11 PTPN11 mutations in the 278 analyzed patients (4.0%, 95% CI 1.7–6.2%), which are listed in Table 1. All of the mutations encode amino-acid substitutions within the N-SH2 domain where it interacts with the PTP domain (Table 1). As in JMML, the majority of mutations are located in exon 3 with 'hotspots' at codons 72 and 76.^{11, 12} One possible limitation of this study is that only nine of the 15 possible exons were screened; however, these are the most commonly mutated exons identified in hematopoietic disorders thus far.

Table 1 - PTPN11 mutations in pediatric AML.

Full table

Clinical characteristics of the study population are summarized in Table 2. There were no differences in diagnostic parameters including the median age, percentage of bone marrow blasts, presence of central nervous system (CNS) disease, hemoglobin, or platelet counts of patients with and without mutations. PTPN11 mutations were more common in boys, but this was not statistically significant (5.9 vs 1.6%, P=0.119). Five of the 11 patients (46%) with PTPN11 mutations had FAB M5 morphology compared to 37/261 (14%) of those without mutations (P=0.016). Taken together, four of the 11 (36%) patients with PTPN11 mutations were boys with FAB M5 morphology. This is significantly higher than the 8% of patients without mutations (P=0.012). In addition, there was a trend for patients with PTPN11 mutations to present with a white blood cell count >100,000 l (P=0.102). The probabilities of achieving a first clinical remission as well as EFS and OS were similar in patients with and without PTPN11 mutations. However, the power to detect significant differences is diminished by the low frequency of mutations.

Table 2 - Clinical and laboratory characteristics of the analyzed cohort.

Full table

These AML samples were also analyzed for FLT3 and NRAS mutations (S Meshinchi et al, Blood 2003; 102: #98 abstract, and S Meshinchi, personal communication, February 2004). One of 11 patients with PTPN11 mutations (9%) had an internal tandem duplication of the FLT3 receptor gene compared with 17% of the rest of the cohort (P=0.808). Whereas the overall incidence of NRAS mutations was 10% in 266 analyzed samples, none of the 11 specimens with PTPN11 mutations showed an NRAS mutation (P=0.608).

PTPN11 mutations are found in 35% of JMML samples;^{11, 12} however, we detected a much lower incidence in de novo pediatric AML (4%). Patients with PTPN11 mutations tend to be boys who present with markedly elevated leukocyte counts and FAB M5 leukemia. Interestingly, the rare cases of AML that evolve from JMML typically show FAB M4 or M5 morphology. The clinical and biologic similarities between JMML and pediatric AML with PTPN11 alterations raise the possibility that both of these myeloid malignancies are initiated by a PTPN11 mutation. Indeed, genetic analysis of human JMML specimens and the phenotype of Kras and Nf1 mutant mice infer that hyperactive Ras can initiate a myeloproliferative disorder.^{20, 21} However, the existing data also support a model in which AML is initiated by genetic lesions leading to aberrant transcription that cooperate with mutations that deregulate signal transduction pathways.^{20, 21} Consistent with this idea, three of the AML patients with PTPN11 mutations also showed recurrent leukemia-associated cytogenetic abnormalities. Other patients may have also demonstrated common cytogenetic abnormalities; however, available data for them were lacking. Rather than acting as an initiating event in AML, we hypothesize that hyperactive SHP-2 proteins favor the outgrowth of malignant cells, perhaps by perturbing signal transduction from specific cytokine receptors.

One interesting clinical observation is that patients with Noonan syndrome have been reported to develop JMML most frequently, followed by reports of acute lymphoblastic leukemia, neuroblastoma, and rhabdomyosarcoma^{22, 23, 24, 25, 26, 27} (Y Aoki et al and A Sarkozy et al, Am Soc Hum Genet 2002; 71: #2096 and #2065, abstracts). They are also at risk of developing transient myeloproliferative syndromes in the neonatal period and coagulopathies.^{28, 29, 30, 31} They do not, however, appear to be at an increased risk of developing AML. While some of the same codons are affected in both NS and AML, many are different. The biochemical effects of these substitutions are not completely understood and it is unclear why patients with NS are not predisposed to developing AML.

The lack of NRAS mutations in pediatric AML specimens with PTPN11 mutations is consistent with the idea that PTPN11 mutations undermine myeloid growth by deregulating Ras. Similarly, PTPN11 mutations are largely restricted to JMML specimens without mutations in either RAS or NF1, which encodes a GTPase activating protein that negatively regulates Ras output. We were unable to analyze primary cells of patients with PTPN11 mutations for constitutive activation of SHP-2. Some, but not all, cultured cell lines engineered to express mutant SHP-2 proteins show hyperactive Ras signaling.^{11, 12, 32} Further studies are required to address how PTPN11 mutations contribute to specific subsets of AML, and to fully characterize the biochemical consequences of leukemia-associated SHP-2 proteins in hematopoietic cells.

References

1. Gilliland DG, Griffin JD. The roles of FLT3 in hematopoiesis and leukemia. Blood 2002; 100: 1532–1542. | Article | PubMed | ISI | ChemPort |
2. Meshinchi S, Stirewalt DL, Alonzo TA, Zhang Q, Sweetser DA, Woods WG et al. Activating mutations of RTK/ras signal transduction pathway in pediatric acute myeloid leukemia. Blood 2003; 102: 1474–1479. | Article | PubMed | ISI | ChemPort |
3. Radich JP, Kopecky KJ, Willman CL, Weick J, Head D, Appelbaum F et al. N-ras mutations in adult de novo acute myelogenous leukemia: prevalence and clinical significance. Blood 1990; 76: 801–807. | PubMed | ISI | ChemPort |
4. Nakao M, Yokota S, Iwai T, Kaneko H, Horiike S, Kashima K et al. Internal tandem duplication of the flt3 gene found in acute myeloid leukemia. Leukemia 1996; 10: 1911–1918. | PubMed | ISI | ChemPort |
5. Hof P, Pluskey S, Dhe-Paganon S, Eck MJ, Shoelson SE. Crystal structure of the tyrosine phosphatase SHP-2. Cell 1998; 92: 441–450. | Article | PubMed | ISI | ChemPort |
6. Tartaglia M, Mehler EL, Goldberg R, Zampino G, Brunner HG, Kremer H et al. Mutations in PTPN11, encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome. Nat Genet 2001; 29: 465–468. | Article | PubMed | ISI | ChemPort |
7. Tartaglia M, Kalidas K, Shaw A, Song X, Musat DL, van der Burgt I et al. PTPN11 mutations in Noonan syndrome: molecular spectrum, genotype-phenotype correlation, and phenotypic heterogeneity. Am J Hum Genet 2002; 70: 1555–1563. | Article | PubMed | ISI | ChemPort |
8. Arico M, Biondi A, Pui CH. Juvenile myelomonocytic leukemia. Blood 1997; 90: 479–488. | PubMed | ChemPort |
9. Emanuel PD, Shannon KM, Castleberry RP. Juvenile myelomonocytic leukemia: molecular understanding and prospects for therapy. Mol Med Today 1996; 2: 468–475. | Article | PubMed | ISI | ChemPort |
10. Niemeyer CM, Arico M, Basso G, Biondi A, Cantu Rajnoldi A, Creutzig U et al. Chronic myelomonocytic leukemia in childhood: a retrospective analysis of 110 cases. European Working Group on Myelodysplastic Syndromes in Childhood (EWOG-MDS). Blood 1997; 89: 3534–3543. | PubMed | ISI | ChemPort |
11. Tartaglia M, Niemeyer CM, Fragale A, Song X, Buechner J, Jung A et al. Somatic mutations in PTPN11 in juvenile myelomonocytic leukemia, myelodysplastic syndromes and acute myeloid leukemia. Nat Genet 2003; 34: 148–150. | Article | PubMed | ISI | ChemPort |
12. Loh ML, Vattikuti S, Schubbert S, Reynolds MG, Carlson E, Lieuw KH et al. Mutations in PTPN11 implicate the SHP-2 phosphatase in leukemogenesis. Blood 2004; 103: 2325–2331. | Article | PubMed | ISI | ChemPort |
13. Sievers EL, Lange BJ, Sondel PM, Krailo MD, Gan J, Liu-Mares W et al. Feasibility, toxicity, and biologic response of interleukin-2 after consolidation chemotherapy for acute myelogenous leukemia: a report from the Children's Cancer Group. J Clin Oncol 1998; 16: 914–919. | PubMed | ISI | ChemPort |
14. Sievers EL, Lange BJ, Sondel PM, Krailo MD, Gan J, Tjoa T et al. Children's cancer group trials of interleukin-2 therapy to prevent relapse of acute myelogenous leukemia. Cancer J Sci Am 2000; 6 (Suppl 1): S39–S44. | PubMed | ISI |
15. Meshinchi S, Woods WG, Stirewalt DL, Sweetser DA, Buckley JD, Tjoa TK et al. Prevalence and prognostic significance of Flt3 internal tandem duplication in pediatric acute myeloid leukemia. Blood 2001; 97: 89–94. | Article | PubMed | ISI | ChemPort |
16. Kaplan EL, Meier P. Nonparametric estimation from incomplete observations. J Am Stat Assoc 1958; 53: 457–481. | Article | ISI |
17. Greenwood M. Reports on Public Health and Medical Subjects, Vol. 33. London: His Majesty's Stationery Office, 1926.
18. Peto R, Peto J. Asymptotically efficient rank in variant test procedures. J R Stat Soc A 1972; 135: 185–206. | ISI |
19. Grimwade D, Walker H, Oliver F, Wheatley K, Harrison C, Harrison G et al. The importance of diagnostic cytogenetics on outcome in AML: analysis of 1,612 patients entered into the MRC AML 10 trial. The Medical Research Council Adult and Children's Leukaemia Working Parties. Blood 1998; 92: 2322–2333. | PubMed | ISI | ChemPort |
20. Braun BS, Tuveson DA, Kong N, Le DT, Kogan SC, Rozmus J et al. Somatic activation of oncogenic Kras in hematopoietic cells initiates a rapidly fatal myeloproliferative disorder. Proc Natl Acad Sci USA. 2004; 101: 597–602. | Article | PubMed | ChemPort |
21. Chan IT, Kutok JL, Williams IR, Cohen S, Kelly L, Shigematsu H et al. Conditional expression of oncogenic K-ras from its endogenous promoter induces a myeloproliferative disease. J Clin Invest 2004; 113: 528–538. | Article | PubMed | ISI | ChemPort |
22. Jung A, Bechthold S, Pfluger T, Renner C, Ehrt O. Orbital rhabdomyosarcoma in Noonan syndrome. J Pediatr Hematol Oncol 2003; 25: 330–332. | Article | PubMed |
23. Agras PI, Baskin E, Sakallioglu AE, Arda IS, Ayter S, Oguzkan S et al. Neurofibromatosis – Noonan's syndrome with associated rhabdomyosarcoma of the urinary bladder in an infant: case report. J Child Neurol 2003; 18: 68–72. | PubMed |
24. Bisogno G, Murgia A, Mammi I, Strafella MS, Carli M. Rhabdomyosarcoma in a patient with cardio-facio-cutaneous syndrome. J Pediatr Hematol Oncol 1999; 21: 424–427. | Article | PubMed |
25. Khan S, McDowell H, Upadhyaya M, Fryer A. Vaginal rhabdomyosarcoma in a patient with Noonan syndrome. J Med Genet 1995; 32: 743–745. | PubMed |
26. Johannes JM, Garcia ER, De Vaan GA, Weening RS. Noonan's syndrome in association with acute leukemia. Pediatr Hematol Oncol 1995; 12: 571–575. | PubMed | ISI | ChemPort |
27. Klopfenstein KJ, Sommer A, Ruymann FB. Neurofibromatosis – Noonan syndrome and acute lymphoblastic leukemia: a report of two cases. J Pediatr Hematol Oncol 1999; 21: 158–160. | Article | PubMed |
28. Bader-Meunier B, Tchernia G, Miélot F, Fontaine JL, Thomas C, Lyonnet S et al. Occurrence of myeloproliferative disorder in patients with the Noonan syndrome. J Pediatr 1997; 130: 885–889. | PubMed | ChemPort |
29. Bertola DR, Carneiro JD, D'Amico EA, Kim CA, Albano LM, Sugayama SM et al. Hematological findings in Noonan syndrome. Rev Hosp Clin Fac Med Sao Paulo 2003; 58: 5–8. | PubMed |
30. Massarano AA, Wood A, Tait RC, Stevens R, Super M. Noonan syndrome: coagulation and clinical aspects. Acta Paediatr 1996; 85: 1181–1185. | PubMed |
31. Witt DR, McGillivray BC, Allanson JE, Hughes HE, Hathaway WE, Zipursky A et al. Bleeding diathesis in Noonan syndrome: a common association. Am J Med Genet 1988; 31: 305–317. | PubMed |
32. Fragale A, Tartaglia M, Wu J, Gelb BD. Noonan syndrome-associated SHP2/PTPN11 mutants cause EGF-dependent prolonged GAB1 binding and sustained ERK2/MAPK1 activation. Hum Mutat 2004; 23: 267–277. | Article | PubMed | ISI | ChemPort |

Acknowledgements

MLL is supported by NIH Grant K23 CA80915, the US Army Chronic Myelogenous Leukemia Program (Project CM020058), the Hope Street Kids Foundation, the UCSF REAC intramural grants program, the Jeffrey and Karen Peterson Family Foundation, and by the Frank A Campini Foundation. Coded specimens from patients with pediatric AML were supplied by the Children's Oncology Group.